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The cytoplasm of yeast contains several enzymes for the reduction of fumarate to succinate. One of these is excluded on Sephadex G-200 and in this respect as in all of its known properties, resembles mitochondrial succinate dehydrogenase in the soluble form. Another type is distinguished by the apparent inability to oxidize succinate with any of the conventional electron acceptors. This enzyme has been subdivided into two types (I and II) which may be readily separated by chromatography on hydroxylapatite. Type I and II fumarate reductases differ from mitochondrial succinate dehydrogenase in containing acid-extractable (rather than covalently bound) FAD, in their intracellular location, in being more stable and more resistant to inhibition by mercurial. The fumarate reductases also differ from the dehydrogenase in molecular weight, in lack of activation by substrate, and in having lower affinities for succinate and malonate and a higher one for fumarate. Conditions which are optimal for the development of mitochondrial succinate dehydrogenase (high O2 tension, low glucose concentration) repress the development of type I and II fumarate reductases and vice versa. ‘Petite’ mutants, which lack succinate dehydrogenase, have a normal content of these fumarate reductases. Type I and II fumarate reductases, while similar in most respects, differ in molecular weight, kinetic constants and reactivity with electron donors. [ABSTRACT FROM AUTHOR]

Autolysates of brewer's yeast contain several isoenzymes of fumarate reductase. One group (Type I) has a molecular weight of 62,000 to 63,000, as determined by chromatography on Sephadex, and 5 components in the group have been detected and partially or completely separated on triethylaminoethyl cellulose. Another group (Type II) consists of at least 2 isoenzymes. The predominant component shows a molecular weight of 34,000 and the minor one 112,000 ± 10,000 on Sephadex. One of the Type I enzymes has been extensively purified until it shows a single component on gradient, centrifugation, although it appears to be heterogeneous on polyacrylamide electrophoresis. The prosthetic group is FAD and, in addition, iron and possibly, copper appear to be present. In contrast to mitochondrial succinate dehydrogenase the FAD is not covalently bound and the non heme iron is not linked to labile sulfide. Preliminary electron paramagnetic resonance data suggest, that the iron may undergo oxidation-reduction during the catalytic cycle. The absorption spectrum manifests the 450 mμ band of flavoproteins but, there is an anomalous absorption below 400 mμ and in the long wave-length region. Dithionite and succinate bleach the 450 mμ band and fumarate partially restores the color of the enzyme. [ABSTRACT FROM AUTHOR]

The effect of various agents on the activation of succinate dehydrogenase in cauliflower (Brassica oleracea) and mung bean (Phaseolus aureus) mitochondria and in sonicated particles has been investigated. Reduced coenzyme Q(10), inosine diphosphate, inosine triphosphate, acid pH, and anions activate the enzyme in mitochondria from higher plants in the same manner as in mammalian preparations. Significant differences have been detected in the behavior of plant and animal preparations in the effects of ATP, ADP, NADH, NAD-linked substrates, and of 2, 4-dinitrophenol on the state of activation of the dehydrogenase. In mammalian mitochondria ATP activates, whereas ADP does not, and the ATP effect is shown only in intact mitochondria. In mung bean and cauliflower mitochondria, both ATP and ADP activate and the effect is also shown in sonicated and frozen-thawed preparations. In sonicated mung bean mitochondria NADH causes complete activation, as in mammalian submitochondrial particles, but in sonicated cauliflower mitochondria activation by NADH is incomplete, as is also true of intact, anaerobic cauliflower mitochondria. Moreover, neither NAD-linked substrates nor a combination of these with NADH can fully activate the enzyme in cauliflower mitochondria. In contrast to mammalian mitochondria, succinate dehydrogenase is not deactivated in cauliflower or mung beam mitochondria under the oxidized conditions brought about by uncoupling of oxidative phosphorylation by 2,4-dinitrophenol.

The spectrophotometric phenazine methosulfate assay of succinate dehydrogenase was adapted to use with cauliflower (Brassica oleracea) and mung bean (Phaseolus aureus) mitochondria with suitable modifications to overcome the permeability barrier to the dye. Procedures in the literature for the isolation and sonic disruption of mitochondria from these sources were modified to assure maximal yield and stability of the enzyme. In tightly coupled mung bean mitochondria, as isolated, about half of the succinate dehydrogenase is in the deactivated state, and the enzyme is further extensively deactivated on sonication or freeze-thawing. In cauliflower mitochondria most of the enzyme is in the deactivated form, and little or no further deactivation occurs on sonication or freeze-thawing. Incubation of mitochondria from either source with succinate leads to full activation of the enzyme. The energy of activation for the conversion of the deactivated to the activated form in membranal preparations under the influence of substrate is about 30,000 cal/mole, essentially the same value as in animal tissues. Activation of the enzyme also occurs under the influence of a variety of other agents, among which the action of anions as activators is documented in the present paper. Activation is accompanied by the release of very tightly bound oxaloacetate. As in animal tissues, the enzyme appears to contain covalently bound flavin (histidyl 8alpha-FAD), and the turnover number is 19,400 moles of succinate oxidized/mole of histidyl flavin at pH 7.5, 38 C.